The present disclosure relates generally to transformed host cells and their use for the production of branched-chain acyl-acyl carrier proteins (acyl-ACP), producing branched-chain fatty acids, and producing branched-chain fatty acyl-ACP chemicals and fuels. More particularly, disclosed herein are transformed host cells and methods for producing branched-chain acyl-ACPs in high titer and high percentages. Also disclosed are methods for producing specific branched-chain fatty acid species and producing branched-chain fatty acid-derived chemicals and fuels such as, for example, branched-chain alcohols and branched-chain fatty acid ethyl esters.
Engineering microbes for the production of advanced biofuels with tunable physical and combustion properties is an attractive response to combat significant global petroleum supply concerns. Extensive research is currently focused on engineering the fatty acid biosynthetic pathway. Fatty acids are common precursors that can be readily converted to several types of chemicals including alkanes, alkenes, alcohols, aldehydes, and esters through either biological or chemical conversion. Bacteria utilize the multienzyme fatty acid synthase II (FASII) platform for fatty acid production, which has been extensively characterized and is widely conserved between organisms, making it an ideal engineering target for fatty acid-derived biofuels with tunable properties. A variety of strategies have been developed to increase titers and yields of fatty acid production in engineered microbes, including directing product formation via substrate supplementation, dynamic regulation of intermediate enzymes, thioesterase variation, and reversal of β-oxidation.
The majority of current efforts have focused on the production of straight-chain fatty acids and SCFA-derived chemicals. Meanwhile, branched chains improve vital fuel properties such as the freezing point, cold flow, and cloud point. Previous work demonstrated the capability to produce BCFA in E. coli through the expression of the branched-chain specific B. subtilis FabH2 in conjunction with BKD and the cytosolic E. coli thioesterase TesA. This, in addition to approaches such as dynamic regulation of FabH and control of product formation through branched-chain amino acids, resulted in production of 20% ante-iso BCFA. Knocking out the straight-chain-specific E. coli FabH and using a variety of branched-chain-specific FabH enzymes and upstream precursors increased the proportion of BCFA, ultimately generating 52% BCFA. Despite these systematic efforts, the predominant products in these studies were SCFAs, which have similar physico-chemical properties and are very difficult to separate from BCFAs. Accordingly, there exists a need for improved methods for BCFA production.